Chlamydia trachomatis (Mouse Pneumonitis Strain) Induces Cardiovascular Pathology following Respiratory Tract Infection

Chlamydia, especially Chlamydia pneumoniae, infection is closely associated with human cardiovascular diseases. Thus far, however, few experimental studies have been carried out to investigate whether natural C. trachomatis infection can induce cardiovascular pathological changes. In this article, we report that pulmonary infection with C. trachomatis mouse pneumonitis strain (MoPn) can induce myocardial and perivascular inflammation and fibrosis in C57BL/6 mice. The pulmonary MoPn infection appeared to be disseminated systemically, because chlamydial antigens were readily detectable in multiple organs including the cardiovascular tissues. In addition, gamma interferon gene knockout mice with a C57BL/6 genetic background showed significant endocarditis and pancarditis characterized by vegetation in aortic valves, interstitial and pericardial inflammatory cellular infiltration, and growth of the organisms in the heart following respiratory tract MoPn infection. The results indicate that C. trachomatis can induce cardiovascular diseases following respiratory tract infection and suggest that murine MoPn respiratory tract infection may be a useful experimental model for investigating cardiovascular diseases caused by chlamydial infection.     

Susceptibility of Chlamydia trachomatis to protegrins and defensins.

We compared the susceptibilities of Chlamydia trachomatis elementary bodies (EBs) to human defensin HNP-2 and porcine protegrin PG-1, cysteine-rich beta-sheet antimicrobial peptides produced by mammalian leukocytes. Although both peptides protected McCoy cell monolayers from infection by chlamydial EBs, protegrins were especially potent. Protegrin-mediated inactivation of chlamydiae occurred rapidly, was relatively independent of the presence of serum, and was effective against serovars L2, D, and H. Protegrin-treated EBs showed striking morphological changes, with obvious damage to their limiting membranes and loss of their cytoplasmic contents and nucleoid. Their effectiveness against chlamydial EBs and other sexually transmitted pathogens combined with their relative lack of cytotoxicity suggests that protegrins and related molecules could serve as prototypes for topical agents to prevent sexually transmitted chlamydial infection.     

Resolution of Chlamydia trachomatis Infection Is Associated with a Distinct T Cell Response Profile

Chlamydia trachomatis is the causative agent of the most frequently reported bacterial sexually transmitted infection, the total burden of which is underestimated due to the asymptomatic nature of the infection. Untreated C. trachomatis infections can cause significant morbidities, including pelvic inflammatory disease and tubal factor infertility (TFI). The human immune response against C. trachomatis, an obligate intracellular bacterium, is poorly characterized but is thought to rely on cell-mediated immunity, with CD4⁺ and CD8⁺ T cells implicated in protection. In this report, we present immune profiling data of subjects enrolled in a multicenter study of C. trachomatis genital infection. CD4⁺ and CD8⁺ T cells from subjects grouped into disease-specific cohorts were screened using a C. trachomatis proteomic library to identify the antigen specificities of recall T cell responses after natural exposure by measuring interferon gamma (IFN-γ) levels. We identified specific T cell responses associated with the resolution of infection, including unique antigens identified in subjects who spontaneously cleared infection and different antigens associated with C. trachomatis-related sequelae, such as TFI. These data suggest that novel and unique C. trachomatis T cell antigens identified in individuals with effective immune responses can be considered as targets for vaccine development, and by excluding antigens associated with deleterious sequelae, immune-mediated pathologies may be circumvented.     

Immunization with the Chlamydia trachomatis Mouse Pneumonitis Major Outer Membrane Protein Can Elicit a Protective Immune Response against a Genital Challenge

Infertility, ectopic pregnancy, and chronic abdominal pain are frequent complications of genital infections with Chlamydia trachomatis. In an attempt to produce a vaccine to protect against this pathogen we purified and refolded the C. trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP). This preparation, mixed with Freund's adjuvant using vortexing or sonication, was used to immunize BALB/c mice that were subsequently challenged in the upper genital tract. Vaginal cultures were taken on a weekly basis, and mice were mated 6 weeks after the challenge. Gels of the vortexed MOMP showed a predominant band with a molecular size of 62 kDa and weaker bands at 42 and 132 kDa, while the sonicated MOMP had a single band with a molecular size of 42 kDa. Following immunization with these two preparations, strong humoral and cell-mediated immune responses were detected in the mice inoculated with the vortexed MOMP. On the other hand, mice immunized with the sonicated MOMP had a strong humoral immune response but a relatively weak cell-mediated immune response, as determined by a T-cell lymphoproliferative assay and level of cytokine production by splenocytes. Vaginal cultures showed that the mice immunized with the vortexed MOMP were significantly protected, as determined by a decrease in the number of animals with positive cultures, the length of time the mice shed viable organisms, and the number of inclusion-forming units recovered per mouse. Animals immunized with the sonicated MOMP, on the other hand, showed a weaker level of protection based on the same three parameters. After mating, the number of fertile animals and number of embryos per mouse were significantly higher for the mice immunized with vortexed MOMP, but not for the mice immunized with sonicated MOMP, compared to those of the control groups. In conclusion, immunization with a purified and refolded preparation of the C. trachomatis MoPn MOMP confers a significant level of protection in mice against a genital challenge.     

Chlamydia trachomatis Persistence in the Female Mouse Genital Tract: Inducible Nitric Oxide Synthase and Infection Outcome

It was previously reported that female mice resolve a primary Chlamydia trachomatis urogenital infection independent of inducible nitric oxide synthase (iNOS). We now report that although iNOS-deficient (NOS2(-/-)) mice resolve culture-apparent infection in a fashion similar to that of normal control (NOS2(+/+)) mice, they sustain significantly increased rates of disease, as assessed by hydrosalpinx formation. PCR amplification of ompA followed by Southern blot detection of amplicands revealed the presence of chlamydial DNA in the lower genital tracts of both NOS2(-/-) and NOS2(+/+) mice at > or =120 days postinfection and in upper genital tract tissues at >120 days postinfection. However, only NOS2(-/-) mice shed low numbers of viable chlamydiae from the lower genital tract after immunosuppressive treatment at 120 days postinfection. When cultured primary murine lung fibroblasts were activated in the presence of gamma interferon (IFN-gamma), inhibition of chlamydial growth occurred in both NOS2(+/+) and NOS2(-/-) cells, but the inhibition was reversible after removal of the cytokine in the NOS2(-/-) primary cell culture only. The iNOS-independent inhibition was microbistatic but was independent of 2,3-indoleamine dioxygenase activity. We conclude that chlamydial DNA and antigens persist in mice subsequent to culture-apparent resolution. In addition, IFN-gamma induces in vivo inhibition of chlamydial growth through microbistatic mechanisms in the absence of iNOS activity, but in the presence of iNOS activity, IFN-gamma is microbicidal and effects eradication.     

Susceptibility to Progressive Cryptococcus neoformans Pulmonary Infection Is Regulated by Loci on Mouse Chromosomes 1 and 9

Genetic factors that regulate the pathogenesis of pneumonia caused by the fungus Cryptococcus neoformans are poorly understood. Through a phenotypic strain survey we observed that inbred C3H/HeN mice develop a significantly greater lung fungal burden than mice of the resistant CBA/J strain 4 weeks following intratracheal infection with C. neoformans ATCC 24067. The aim of the present study was to characterize the inflammatory response of C3H/HeN mice following C. neoformans pulmonary infection and to identify genetic loci that regulate host defense. Following cryptococcal infection, C3H/HeN mice demonstrated a Th2 immune response with heightened airway and tissue eosinophilia, goblet cell metaplasia, and significantly higher lung interleukin-5 (IL-5) and IL-13 protein expression relative to CBA/J mice. Conversely, CBA/J mice exhibited greater airway and tissue neutrophilia that was associated with significantly higher pulmonary expression of gamma interferon, CXCL10, and IL-17 proteins than C3H/HeN mice. Using the fungal burden at 4 weeks postinfection as a phenotype, genome-wide quantitative trait locus (QTL) analysis among 435 segregating (C3H/HeN × CBA/J)F2 (C3HCBAF2) hybrids identified two significant QTLs on chromosomes 1 (Cnes4) and 9 (Cnes5) that control susceptibility to cryptococcal pneumonia in an additive manner. Susceptible C3H/HeN mice carry a resistance allele at Cnes4 and a susceptibility allele at Cnes5. These studies reveal additional genetic complexity of the host response to C. neoformans that is associated with divergent patterns of pulmonary inflammation.     

Plasmid CDS5 Influences Infectivity and Virulence in a Mouse Model of Chlamydia trachomatis Urogenital Infection

The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene.     

Gamma Interferon Production by Cytotoxic T Lymphocytes Is Required for Resolution of Chlamydia trachomatis Infection

In this study, we used mice in which the gene for gamma interferon (IFN-γ) has been disrupted (IFN-γ^−/− mice) to study the role of this cytokine in the resolution of Chlamydia trachomatis infection. We show that IFN-γ^−/− mice are impaired in the ability to clear infection with C. trachomatis compared to IFN-γ^+/+ control mice. Activated CD8⁺ cytotoxic T lymphocytes (CTL) secrete IFN-γ in response to intracellular infection, and we have shown previously that a Chlamydia-specific CTL line can reduce C. trachomatis infection when adoptively transferred into infected mice. In the present study, we found that when these IFN-γ^+/+ CTL lines are transferred into Chlamydia-infected IFN-γ^−/− mice, the transferred CTL cannot overcome the immune defect seen in the IFN-γ^−/− mice. We also show that Chlamydia-specific CTL can be cultured from IFN-γ-deficient mice infected with C. trachomatis; however, the adoptive transfer of IFN-γ^−/− CTL into infected IFN-γ^+/+ mice does not reduce the level of infection. These results suggest that IFN-γ production by CTL is not sufficient to overcome the defect that IFN-γ^−/− mice have in the resolution of Chlamydia infection, yet IFN-γ production by CTL is required for the protective effect seen upon adoptive transfer of CTL into IFN-γ^+/+ mice.     

Susceptibility of Fertilized Mouse Eggs to Minute Virus of Mice

Fertilized two-celled mouse eggs deprived of their zona pellucida were susceptible to infection with minute virus of mice. The virus had no deleterious effect on development of embryos cultivated in vitro.     

Decreased Susceptibility to Listeria monocytogenes in Mice After Infection with Trichinella spiralis

Mice challenged intravenously with Listeria monocytogenes at 7 or 21 days after Trichinella spiralis infection demonstrated a higher mean lethal dose and longer survival time than normal mice. This effect of helminthic infection was abrogated after 49 days.     

Increased Susceptibility of Melanin-Concentrating Hormone-Deficient Mice to Infection with Salmonella enterica Serovar Typhimurium

Melanin-concentrating hormone (MCH) was initially identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, the wide distribution of MCH receptors in peripheral tissues suggests additional functions for MCH which remain largely unknown. We have previously reported that mice lacking MCH develop attenuated intestinal inflammation when exposed to Clostridium difficile toxin A. To further characterize the role of MCH in host defense mechanisms against intestinal pathogens, Salmonella enterocolitis (using Salmonella enterica serovar Typhimurium) was induced in MCH-deficient mice and their wild-type littermates. In the absence of MCH, infected mice had increased mortality associated with higher bacterial loads in blood, liver, and spleen. Moreover, the knockout mice developed more-severe intestinal inflammation, based on epithelial damage, immune cell infiltrates, and local and systemic cytokine levels. Paradoxically, these enhanced inflammatory responses in the MCH knockout mice were associated with disproportionally lower levels of macrophages infiltrating the intestine. Hence, we investigated potential direct effects of MCH on monocyte/macrophage functions critical for defense against intestinal pathogens. Using RAW 264.7 mouse monocytic cells, which express endogenous MCH receptor, we found that treatment with MCH enhanced the phagocytic capacity of these cells. Taken together, these findings reveal a previously unappreciated role for MCH in host-bacterial interactions.     

Infection with a plasmid-free variant Chlamydia related to Chlamydia trachomatis identified by using multiple assays for nucleic acid detection.

Clinical samples in transport media from 40 patients exhibiting pathologies potentially caused by Chlamydia trachomatis infection were analyzed for chlamydial nucleic acid, and the results were compared with those of culture. Chlamydial culture was performed by a shell vial centrifugation method with HeLa 229 host cells. Polymerase chain reaction (PCR) assays were used to detect either regions on a 7.5-kb plasmid characteristic of C. trachomatis (plasmid-PCR) or a segment of the 16S rRNA genes (rRNA-PCR). All PCR results were confirmed by hybridization with probes for the specific amplified products in either a Southern or a dot blot format. An RNase protection (RNP) assay was used to detect genus-specific chlamydial 16S rRNA directly from the clinical samples. The PCR assays detected C. trachomatis but not other bacteria, including Chlamydia spp. C. trachomatis was isolated from six samples which were positive by the rDNA-PCR and plasmid-PCR assays. Five of the culture-positive specimens were positive by the RNP assay. Twenty-two samples were negative by all criteria. Surprisingly, nine samples were positive by rRNA-PCR and RNP assays only. Nucleic acid sequencing of the rRNA-PCR-amplified products indicated a close relationship between the variants and C. trachomatis. The data may indicate an unrecognized process in C. trachomatis infection or that these patients were infected by a variant strain of C. trachomatis which lacks the C. trachomatis-specific plasmid.     

Route of Infection That Induces a High Intensity of Gamma Interferon-Secreting T Cells in the Genital Tract Produces Optimal Protection against Chlamydia trachomatis Infection in Mice

The induction of local T helper type 1 (Th1)-mediated cellular immunity is crucial for resistance of mice to genital infection by the obligate intracellular bacterium Chlamydia trachomatis. We tested the hypothesis that the route of immunization that elicits relatively high numbers of chlamydia-specific, gamma interferon (IFN-γ)-secreting T lymphocytes (ISTLs) in the genital tract would induce optimal protective immunity against reinfection. Female BALB/c mice were infected intravaginally (i.v.), intranasally (i.n.), orally (p.o.), or subcutaneously (s.c.) with C. trachomatis. At days 7, 14, 21, and 28 postinfection, T cells isolated from the genital tract tissues were restimulated with chlamydial antigen in vitro, and the amounts of IFN-γ induced were measured by a sandwiched enzyme-linked immunosorbent assay method. At day 7 postinfection, i.n.- and i.v.-immunized mice had high levels of chlamydia-specific ISTLs in their genital tracts (203.58 ± 68.1 and 225.5 ± 12.1 pg/ml, respectively). However, there were no detectable ISTLs in the genital tracts of p.o.- or s.c.-infected mice. When preinfected mice were challenged i.v. 70 days later, animals preexposed by the i.n. route were highly resistant to reinfection, with greatly reduced chlamydial burden, and suffered an attenuated infection that resolved by day 6 postchallenge. Animals preexposed by the i.v. route were modestly protected, whereas p.o. and s.c. groups were indistinguishable in this regard from control mice. The resistance of i.n.-immunized mice (and to some extent the i.v.-exposed mice) to reinfection was associated with early appearance (within 24 h) of high levels of genital ISTLs compared with mice preinfected by other routes. Furthermore, although i.n. and i.v.-immunized mice had comparable levels of chlamydia-specific immunoglobulin A (IgA) antibodies in their vaginal washes, the levels of IgG2a were four- sixfold higher in i.n.-immunized mice than in any of the other groups. The results suggested that immunization routes that foster rapid induction of vigorous genital mucosal cell-mediated immune (CMI) effectors (e.g., IFN-γ), the CMI-associated humoral effector, IgG2a, and to some extent secretory IgA produce protective immunity against chlamydial genital infection. Therefore, i.n. immunization is a potential delivery route of choice in the development of a vaccine against Chlamydia.     

Chlamydia trachomatis IncA Is Localized to the Inclusion Membrane and Is Recognized by Antisera from Infected Humans and Primates

Chlamydia psittaci produces a collection of proteins, termed IncA, IncB, and IncC, that are localized to the chlamydial inclusion membrane. In this report we demonstrate that IncA is also produced by Chlamydia trachomatis. C. trachomatis IncA is structurally similar to C. psittaci IncA and is also localized to the inclusion membrane. Immunoblot analysis demonstrated that sera from C. trachomatis-infected patients and from experimentally infected monkeys both recognized C. trachomatis IncA.     

The Genetic Background Modulates the Effect of the Beige Gene on Susceptibility to Cytomegalovirus Infection in Mice

Homozygous beige (bg/bg) mice were more susceptible to the development of fatal disease induced by murine cytomegalovirus (MCMV) than their bg/ + littermates. However, the increase in susceptibility depended on the genetic background of the strain carrying the bg gene. C57BL/6, SB/Le, DBA/2, and CBA bg/bg mice showed, respectively, 2.5-, 3.2-, 9.5-, and 18.6-fold increases in susceptibility compared with the corresponding bg/+ animals. Beige mice showed higher liver titres of MCMV than bg/ + by the 2nd or 3rd day after infection, and tissue damage was also greater. Splenic NK cells were not detected in uninfected bg/bg mice, and after virus inoculation the increment in cytotoxicity was greater in bg/ + than in bg/bg mice. However, cytotoxicity towards WEHI-164 cells was not impaired in bg/bg mice and was not augmented by MCMV. Interferon titres were also not impaired by the beige mutation. Of the strains examined, CBA had the highest endogenous levels of NK cells and were most genetically resistant to MCMV. Thus, our observation that the beige gene had the greatest effect on susceptibility in this strain suggests that NK cells are important mediators of genetically determined resistance to MCMV.     

Protection against Ascending Infection of the Genital Tract by Chlamydia trachomatis Is Associated with Recruitment of Major Histocompatibility Complex Class II Antigen-Presenting Cells into Uterine Tissue

A mouse model of ascending infection following intravaginal inoculation with a strain of Chlamydia trachomatis isolated from humans has been used to identify immune mechanisms associated with protection against genital infection. BALB/c and C3H mice differed in their susceptibilities to infection and inflammatory disease. In both mouse strains, ascension of the organism and recruitment of bone marrow-derived mononuclear leukocytes were evident in uterine tissue 1 week postinfection. By 3 weeks the organism had been cleared and inflammation had been resolved in the BALB/c mice, but both persisted in the C3H animals. In athymic nude BALB/c mice both the organism and inflammation persisted, indicating the influence of the hosts’ immune response on the outcome of infection. Both BALB/c and C3H mice had a Th1 response in draining lymph nodes, with predominant production of gamma interferon and tumor necrosis factor alpha, low levels of interleukin-10, and no detectable levels of interleukin-4. However, the composition of the early uterine infiltrate differed in these two mouse strains. Cell surface labeling and analysis of light scatter properties by flow cytometry identified a population of large, CD45⁺ major histocompatibility complex class II mononuclear cells, which were a prominent feature of the infiltrates in BALB/c mice but were present in significantly lower numbers in C3H mice. These cells expressed the costimulatory molecules CD86 and CD40 and stimulated allogeneic T cells, suggesting that these mononuclear cells are a population of antigen-presenting cells and that they may play a role in clearing antigen and protecting against inflammatory disease in BALB/c mice. An additional level of immunological control may thus exist in genital chlamydial infection.Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which selectively colonizes epithelial cells in the human host. Infection of the genital tract with C. trachomatis serovars D through K is a major cause of sexually transmitted disease worldwide. Infection is insidious and, though often asymptomatic, can have serious consequences particularly for women. In some cases of cervical infection the organism ascends into the upper genital tract; this is a major cause of pelvic inflammatory disease with sequelae that include infertility and ectopic pregnancy (21).Left untreated, genital chlamydial infections are chronic, and repeated infections are common, indicating that the natural immune response is poorly protective. However, the incidence of genital chlamydial infection falls with increasing age; this might be due to cumulative serovar-specific immunity mediated by a local antibody (3). Antibodies can neutralize infectivity in vitro and in vivo (40) but have not been identified as the dominant protective mechanism in animal studies; antibodies play little part in protection against primary infections (12, 26, 31), although they can protect against severe pathology (7) and can play a subsidiary role in defense against reinfection (31).A predominant role for Th1 CD4⁺ T cells and the production of gamma interferon (IFN-γ) in controlling primary genital infection and preventing spread to other tissues has been implicated in cell transfer studies, antibody-mediated depletion experiments, and infections in gene knockout mice (8, 12, 15, 19, 30). However, in the absence of a functioning IFN-γ system a poorly defined compensatory mechanism can operate (12, 37) and a mechanism dependent on interleukin-12 (IL-12) but independent of IFN-γ may thus be important in the early stages of infection (25). IFN-γ is less important in protection against reinfection than against a primary challenge (8, 37). Thus, a successful immune response against chlamydial infection is flexible and complex, with different mechanisms involved as the infection progresses.Studies of infection in mouse strains with different susceptibilities to disease have proved useful in identifying protective immune mechanisms. We have identified mouse strain differences in disease susceptibility following intrauterine (i.u.) injection with a serovar-F strain of C. trachomatis from humans; C3H mice developed severe disease with prolonged salpingitis resulting in infertility, whereas BALB/c suffered less-severe inflammatory changes and remained fertile (32). Because direct injection of the organism into the upper genital tract did not allow us to distinguish between increased susceptibility to infection and sensitivity to pathological reactions, we developed a model of ascending infection following intravaginal (i.vag.) inoculation of this strain of C. trachomatis. Ascension of the organism into the uterus and oviducts of C3H mice was detected in association with inflammatory changes in genital tract tissue (29).In the present study we compare ascending infections in C3H and BALB/c mice and report increased susceptibility in C3H mice despite the development of a predominantly Th1 response and the production of IFN-γ in the lymph nodes of both mouse strains. Instead, clearance of the organism and protection against inflammatory disease appeared to be associated with recruitment of major histocompatibility complex (MHC) class II antigen-presenting cells (APC) into uterine tissue early in infection. These cells may possibly play a role in the defense against genital chlamydial infection.